Virus strains

ABSTRACT

The present invention relates to non-laboratory virus strains, for example of herpes viruses such as HSV, with improved oncolytic and/or gene delivery capabilities as compared to laboratory virus strains.

FIELD OF THE INVENTION

[0001] The present invention relates to non-laboratory virus strains,for example of herpes viruses such as HSV, with improved oncolyticand/or gene delivery capabilities as compared to laboratory virusstrains.

BACKGROUND OF THE INVENTION

[0002] Viruses have been suggested or demonstrated to have utility in avariety of applications in biotechnology and medicine on many occasions.Each is due to the unique ability of viruses to enter cells at highefficiency. This is followed in such applications by either virus geneexpression and replication and/or expression of an inserted heterologousgene. Thus viruses can either deliver and express genes in cells (eitherviral or other genes) which may be useful in for example gene therapy orthe development of vaccines, or they may be useful in selectivelykilling cells by lytic replication or the action of a delivered gene infor example cancer.

[0003] Herpes simplex virus (HSV) has been suggested to be of use bothas a gene delivery vector in the nervous system and elsewhere and forthe oncolytic treatment of cancer. In both applications the virus musthowever be disabled such that it is no longer pathogenic but such thatit can still enter cells and perform the desired function. Thus fornon-toxic gene delivery to target cells using HSV it has become apparentthat in most cases immediate early gene expression must beprevented/minimised from the virus. For the oncolytic treatment ofcancer, which may also include the delivery of gene(s) enhancing thetherapeutic effect, a number of mutations to HSV have been identifiedwhich still allow the virus to replicate in culture or in activelydividing cells in vivo (e.g. in tumours), but which prevent significantreplication in normal tissue. Such mutations include disruption of thegenes encoding ICP34.5, ICP6 and thymidine kinase. Of these, viruseswith mutations to ICP34.5, or ICP34.5 together with mutations of e.g.ICP6 have so far shown the most favourable safety profile. Virusesdeleted for only ICP34.5 have been shown to replicate in many tumourcell types in vitro and to selectively replicate in artificially inducedbrain tumours in mice while sparing surrounding tissue. Early stageclinical trials have also shown their safety in man.

[0004] However, while promise has been shown for various virusesincluding HSV for gene delivery/therapy or for the oncolytic treatmentof cancer, the majority of this work has used virus strains which havebeen maintained in tissue culture cells for many years. In applicationswhere the virus merely needs enter cells to deliver genes this may notprove problematical as maintenance in cell culture also requires thevirus to enter cells, albeit often cells of a different type or speciesin comparison to the likely target cells for a vector. However, inapplications where other properties are required, the use of laboratoryvirus strains may not allow the full potential of a virus in aparticular application to be utilised.

[0005] HSV has the unique ability amongst viruses currently underdevelopment as vectors in that it has naturally evolved to infect andremain latent in neurons. HSV has also evolved to be highly efficientlytransported along nerves from the site of infection, usually at theperiphery, to the neuronal cell body, usually in the spinal ganglia.Such capabilities are not required in cell culture and as suchcapabilities require specific evolved properties of HSV, furtheradaption to growth in culture may have resulted in optimally efficientaxonal transport capabilities to have been lost. HSV vectors for genedelivery to the central or peripheral nervous system are likely to showmaximum effectiveness if axonal transport properties have been retainedat maximum efficiency. Here, inoculation at a peripheral site would thenallow maximally efficient gene delivery to peripheral neuron cellbodies, and inoculation in the brain would allow maximally efficientgene delivery to multiple connected sites. Current vectors based onlaboratory strains of HSV may not allow this to occur at the maximumefficiency possible. Indeed, because of HSV's high capacity to betransported along nerves, there is potentially a particularly largediscrepancy between the properties which it is desired to conserve andthose likely to be retained in culture.

[0006] HSV and other viruses such as adeno- or rheovirus also havepotential utility in the oncolytic treatment of cancer. However, againviruses under development for such purposes have previously beenextensively maintained in culture. As the oncolytic treatment of cancerrequires active replication in often relatively slowly growing humantumour cells, it would be anticipated that adaptation of laboratoryvirus strains to growth in particular cultured cells may have reducedthe efficiency with which such lytic replication in human tumour cells,or infection of human tumour cells, could optimally occur.

SUMMARY OF THE INVENTION

[0007] The present invention provides the opportunity to develop viruseswith improved in vivo capabilities of gene transfer and/or lyticdestruction of tumour cells. Here, virus strains are constructedappropriate for these purposes based on recent clinical isolates of theappropriate virus rather than the serially passaged laboratory strainswhich have previously been used. The present invention therefore has thepotential to provide viruses with improved capabilities of infectinghuman cells in vivo, improved replicative/lytic capability in suchcells, and (in the case of HSV) improved abilities of trafficking alongnerves from the inoculation site to the neuronal cell body. Theinvention is exemplified using HSV but is equally applicable for otherviruses currently under development as vectors and/or for the oncolyticdestruction of cancer cells.

[0008] We have shown that two clinical isolates of HSV1 (strains JS1 andBL1) have enhanced replication in some human tumour cell lines ascompared to HSV1 strain 17+ (a standard laboratory strain).

[0009] We have deleted ICP34.5 from the clinical isolate JS1 strain andagain compared replicative potential in human tumour cell types incomparison to HSV1 strain 17+ (a standard laboratory strain) in whichICP34.5 was also deleted. This strain (JS1/ICP34.5−) is a modifiedstrain derived from a clinical isolate, and is thus a modifiednon-laboratory strain of the invention.

[0010] JS1 with ICP34.5 deleted showed enhanced growth in some humantumour cells tested as compared to HSV1 ICP34.5 deleted strain 17+, i.e.a laboratory strain virus with the same modification. However, ascompared to the laboratory strain derived from strain 17+, cell killingcapabilities were enhanced with the JS1/ICP34.5− virus in all tumourcell lines tested.

[0011] Thus, the use of non-laboratory virus strains can be seen toenhance the anti-tumour capabilities of such viruses and was evident inall the tumour cell lines tested so far. This will have applicabilityfor cancer treatment in human patients.

[0012] Further enhanced activity may also be anticipated if theseviruses are then used to deliver genes with anti-tumour activity. Suchgenes include those encoding pro-drug activators, tumour suppressor orpro-apoptotic factors, or immune stimulatory proteins.

[0013] For this purpose, we have produced an ICP34.5 deleted clinicalisolate of HSV1 which expresses human GMCSF. This virus is designed toenhance anti-tumour immune responses following intra-tumoral injection.

[0014] The invention also provides viruses of the invention which carrya heterologous gene/genes. The term heterologous gene is intended toembrace any gene not found in the viral genome. The heterologous genemay be any allelic variant of a wild-type gene, or it may be a mutantgene. Heterologous genes are preferably operably linked to a controlsequence permitting expression of said heterologous gene in a cell invivo. Viruses of the invention may thus be used to deliver aheterologous gene/genes to a cell in vivo where it will be expressed.For oncolytic virus therapy, such genes typically encode proteinscapable of enhancing the tumour destroying properties of the virus.These genes may encode proteins which are themselves cytotoxic, arepro-drug activating, or which may be capable of stimulating/enhancing ananti-tumour immune response. For gene delivery to the peripheral nervoussystem using HSV, the heterologous gene/genes may encode a polypeptidecapable of modifying responses to painful stimuli or reducing chronicpain, for example a protein capable sequestering e.g. nerve growthfactor, other pain modulating neurotrophic factor or neurotrophicfactor-like molecules, or substance P or other neuropeptides. Theheterologous gene/genes may also encode a polypeptide capable ofstimulating the re-growth of damage nerves or preventing the furtherdegeneration of nerves in degenerative conditions. In the centralnervous system, heterologous genes may include those potentiallybeneficial in neurodegenerative disease such as Parkinson's disease orAlzheimer's disease and might typically include genes encodingneurotrophic factors and/or enzymes capable of enhancing the activity ofremaining cells in such diseases. In all cases, single or multipleheterologous genes may be carried by a single virus.

[0015] Accordingly the invention provides:

[0016] Use of a modified, oncolytic, non-laboratory virus strain in themanufacture of a medicament for the oncolytic treatment of cancer;

[0017] Use of a modified, replication incompetent, non-laboratory virusstrain comprising a heterologous gene in the manufacture of a medicamentfor the delivery of said gene to a subject;

[0018] A method of determining whether a gene has an effect on aphenotype associated with a peripheral nervous system disorder or on acell of the peripheral nervous system which is relevant to a peripheralnervous system disorder, which method comprises:

[0019] (i) inoculating a replication incompetent herpes virus of theinvention comprising a heterologous gene into a peripheral nerve; and

[0020] (ii) monitoring a phenotype of said disorder or an effect ofexpression of said gene on said cell to determine thereby whether saidgene has an effect relevant to said disorder;

[0021] A method of determining whether a gene has an effect on aphenotype associated with a central nervous system disorder or on a cellof the central nervous system which is relevant to a central nervoussystem disorder, which method comprises:

[0022] (i) inoculating into a cell of the central nervous system with areplication incompetent herpes virus of the invention; and

[0023] (ii) monitoring a phenotype of said disorder or an effect ofexpression of said gene on said cell to determine thereby whether saidgene has an effect on said cell or said phenotype;

[0024] A method of determining whether a gene encodes an antigenassociated with a pathogenic infection or cancer, which method comprisesinfecting a dendritic cell or a macrophage with a replicationincompetent virus of the invention comprising a heterologous geneencoding an antigen and monitoring antigen presentation of thepolypeptide product of said gene, or an effect of expression of saidgene, or a phenotype of said pathogenic infection or cancer to determinethereby whether said gene encodes an antigen associated with saidinfection or cancer and which itself has therapeutic potential or is atarget for therapeutic intervention;

[0025] A method of determining the suitability of a non-laboratory virusstrain for modification into a modified strain as defined herein,comprising:

[0026] (i) optionally, isolating a non-laboratory virus strain from ahost;

[0027] (ii) providing said non-laboratory virus strain;

[0028] (iii) assessing the properties of the virus in respect of one ormore desirable characteristics; and, optionally,

[0029] (iv) selecting for modification virus strains with desirableproperties;

[0030] A method of determining the suitability of a non-laboratory virusstrain for modification into a modified oncolytic strain of theinvention, comprising:

[0031] (i) optionally, isolating a non-laboratory virus strain from ahost;

[0032] (ii) assessing the growth of the virus in one or more types oftumour cell; and optionally

[0033] (iii) selecting for modification virus strains with a high growthrate or cell killing capability;

[0034] A method of determining the suitability of a non-laboratory virusstrain for modification into a modified oncolytic strain, comprising:

[0035] (i) providing a non-laboratory virus strain, optionally oneselected by a method as just defined;

[0036] (ii) modifying said strain such that it becomes oncolytic; and

[0037] (iii) assessing the ability of said modified, oncolyticnon-laboratory strain to kill tumour cells; and optionally

[0038] (iv) selecting strains that exhibit high tumour cell-killingcapacity for further modifications; and optionally

[0039] (v) carrying out further modifications;

[0040] A method of determining whether a gene enhances the anti-tumoureffects of a virus comprising:

[0041] (i) providing a modified, oncolytic non-laboratory strain of theinvention;

[0042] (ii) inserting said gene into said virus as a heterologous gene;and

[0043] (iii) assessing the ability of said modified, oncolyticnon-laboratory strain to kill tumour cells compared to the ability ofthe precursor strain provided in step (i);

[0044] A method of producing a modified, oncolytic, non-laboratory virusstrain comprising:

[0045] (i) isolating from a host a non-laboratory strain of a virus;

[0046] (ii) optionally determining its suitability for modification asdefined above; and

[0047] (iii) modifying it to render it oncolytic, and optionally

[0048] (iv) carrying out further modifications;

[0049] A method of producing a modified non-laboratory virus straincomprising:

[0050] (i) providing a non-laboratory strain of a virus;

[0051] (ii) modifying it to render it replication incompetent and,

[0052] (iii) inserting a heterologous gene;

[0053] A modified, oncolytic, non-laboratory virus strain as definedherein;

[0054] A modified non-laboratory virus strain comprising a heterologousgene, as defined herein

[0055] Use of a gene identified as having an effect on a phenotypeassociated with a peripheral nervous system disorder or on a cell of theperipheral nervous system which is relevant to a peripheral nervoussystem disorder by a method as defined above, or of a gene productencoded by said gene in the manufacture of a medicament for thetreatment of a peripheral nervous system disorder;

[0056] Use of a gene identified as having an effect on a phenotypeassociated with a central nervous system disorder or on a cell of thecentral nervous system which is relevant to a central nervous systemdisorder by a method as defined above, or of a gene product encoded bysaid gene in the manufacture of a medicament for the treatment of acentral nervous system disorder;

[0057] Use of a gene identified as encoding an antigen associated with apathogenic infection or cancer by a method as defined above, or of anantigen encoded by said gene in the manufacture of a medicament for thetreatment or prevention of said infection or cancer;

[0058] A non-laboratory virus strain identified by, or produced in thecourse of, a method of the invention;

[0059] Use of a gene identified as enhancing the anti-tumour effects ofa virus by a method as defined above, or of a gene product encoded bysaid gene, in the manufacture of a medicament for the treatment orprevention of cancer;

[0060] A modified non-laboratory virus strain obtained or obtainable bya method of the invention;

[0061] HSV1 strain JS1 as deposited at the European Collection of CellCultures (ECACC) under provisional accession number 01010209, or an HSV1strain derived therefrom, a pharmaceutical composition comprising such avirus; such a virus for use in the treatment of the human or animalbody;

[0062] A method of treating a tumour in an individual in need thereof byadministering to said individual an effective amount of an oncolyticvirus of the invention;

[0063] A method of delivering a gene to an individual in need thereof byadministering to said individual an effective amount of a non-oncolyticvirus of the invention; and

[0064] A method of treating or preventing a central peripheral nervoussystem disorder by administering to a peripheral nerve of an individualin need thereof an effective amount of a neurotrophic virus of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065]FIG. 1. Viruses

[0066] From top to bottom, diagrams show: laboratory HSV1 strain 17+,clinical strain BL1, clinical strain JS1, 17+/ICP34.5−, JS1 /ICP34.5−,JS1/ICP34.5−/ICP47−/hGMCSF.

[0067]FIG. 2. Clinical isolates show enhanced growth in tumour cells

[0068] (1) Growth of 17+, BL1 and JS1. Left hand diagram: U87 cells.Right-hand diagram: LNCaP cells.

[0069] (2) Growth of ICP34.5− 17+ and JS1 on tumour cells. Left-handdiagram: LNCaP cells. Right-hand diagram: MDA-MB-231 cells.

[0070] (3) JS1/34.5− does not grow on cells non-permissive for HSVICP34.5 mutants. Left-hand diagram: 3T6 cells—17+, JS1. Right-handdiagram: 3T6 cells—17+, JS1 ICP34.5−.

[0071]FIG. 3. An ICP34.5 deleted HSV clinical isolate shows enhancedlysis in all tumour cells tested

[0072] Tumour cell lines were either mock infected, infected with HSV1strain 17+/34.5−, or infected with HSV1 strain JS3/34.5− at theindicated MOI and stained with crystal violet at time points afterinfection to allow visualisation of cells. Each block of photographsrelates to a cell type. From top to bottom, these are HT29 colorectaladenocarcinoma, LNCaP.FGC prostate adenocarcinoma, MDA-MB-231 breastadenocarcinoma, SK-MEL-28 malignant melanoma and U-87 MG glioblastomaastrocytoma. Left-hand blocks relate to results for HSV1 strain17+/34.5−. Right-hand blocks relate to results for HSV1 strainJS1/34.5−. Central blocks represent mock infected cells. Within eachblock, the top row represents a 24 hour time-point, the second a 48 hourtime-point and the third a 72 hour time-point within each block, theleft-hand column represents MOI=0.2, the central column MOI=0.1 and theright-hand column MOI=5.

DETAILED DESCRIPTION OF THE INVENTION

[0073] A. Viruses

[0074] Virus Strains of the Invention

[0075] The invention is applicable to viruses in general. Preferably, avirus strain of the invention will be a strain of a herpes virus,adenovirus, picomavirus, retrovirus or alphavirus. More preferably, avirus strain of the invention will be a strain of a herpes virus. Stillmore preferably, a virus strain of the invention will be a strain of aherpes simplex virus (HSV), typically a strain of HSV1 or HSV2.

[0076] When the virus of the invention is a herpes simplex virus, thevirus may be derived from, for example HSV1 or HSV2 strains, orderivatives thereof, preferably HSV1. Derivatives include inter-typerecombinants containing DNA from HSV1 and HSV2 strains. Such inter-typerecombinants are described in the art, for example in Thompson et al(1998) and Meignier et al (1988). Derivatives preferably have at least70% sequence homology to either the HSV1 or HSV2 genomes, morepreferably at least 80%, even more preferably at least 90 or 95%. Morepreferably, a derivative has at least 70% sequence identity to eitherthe HSV1 or HSV2 genome, more preferably at least 80% identity, evenmore preferably at least 90%, 95% or 98% identity.

[0077] For example the UWGCG Package provides the BESTFIT program whichcan be used to calculate homology (for example used on its defaultsettings) (Devereux et al. (1984) Nucleic Acids Research 12, p387-395).The PILEUP and BLAST algorithms can be used to calculate homology orline up sequences (typically on their default settings), for example asdescribed in Altschul (1993) J. Mol. Evol. 36:290-300; Altschul et al.(1990) J. Mol. Biol. 215:403-10.

[0078] Software for performing BLAST analyses is publicly availablethrough the National Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al., 1990). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score falls off by the quantity X fromits maximum achieved value; the cumulative score goes to zero or below,due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both strands.

[0079] The BLAST algorithm performs a statistical analysis of thesimilarity between two sequences; see e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a sequence is considered similar to another sequence if thesmallest sum probability in comparison of the first sequence to thesecond sequence is less than about 1, preferably less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

[0080] A derivative may have the sequence of a HSV1 or HSV2 genomemodified by nucleotide substitutions, for example from 1, 2 or 3 to 10,25, 50 or 100 substitutions. The HSV1 or HSV2 genome may alternativelyor additionally be modified by one or more insertions and/or deletionsand/or by an extension at either or both ends.

[0081] Properties of Virus Strain of the Invention

[0082] Virus strains of the invention are “non-laboratory” strains. Theycan also be referred to as “clinical” strains. A person of skill in theart will readily be able to distinguish between a laboratory strain anda non-laboratory, or clinical, one. Further guidance on the propertieslikely to be exhibited by virus strains is given below.

[0083] The key distinction between a laboratory and non-laboratorystrain is that laboratory strains currently in common use have beenmaintained for long periods, many years in some cases, in culture. Theculture of viruses such as HSV involves a technique known as serialpassage. To grow and maintain viruses, suitable cells are infected withthe virus, the virus replicates within the cell and the virus is thenharvested; fresh cells are then re-infected. this process constitutesone cycle of serial passage. Each such cycle may take, for example, afew days in the case of HSV. As discussed above, such serial passagingmay lead to changes in the properties of the virus strain, in thatselection takes places for properties that favour growth in culture(e.g. rapid replication), as opposed to properties useful for practicalapplications, e.g. maintenance of the capacity to travel along axons inthe case of HSV.

[0084] Virus strains of the invention are non-laboratory strains in thatthey are derived from strains recently isolated from infectedindividuals. Strains of the invention are modified compared to theoriginal clinical isolates, and may have spent a time in culture, butany time spent in culture will be comparatively short. Strains of theinvention are prepared in such a manner as to retain substantially thedesirable properties of the original clinical isolates from which theyare derived.

[0085] A virus of the invention is capable of efficiently infectingtarget human cells. Such a virus is recently isolated from an infectedindividual and then screened for the desired ability of enhancedreplication in tumour and/or other cells in vitro and/or in vivo incomparison to standard laboratory strains, or (in the case ofneurotrophic viruses such as HSV) for an enhanced ability to trafficalong nerves as compared to standard laboratory strains using an in vivomodel. Such viruses with improved properties as compared to laboratoryvirus strains are viruses of the invention. Identified viruses with suchdesired improved properties can then be engineered such that they canselectively kill tumour cells by the mutation of appropriate gene(s), ormutated such that they can deliver a gene(s) to target tissue withouttoxic effect in non-oncolytic applications. These modified viruses arealso viruses of the invention. Alternatively, virus strains may beisolated from an infected individual and mutations anticipated to beappropriate for oncolytic therapy and/or gene delivery made. Thesemodified viruses are then screened for the desired improved propertiesas compared to laboratory strains, viruses with such improved propertiesproviding further viruses of the invention.

[0086] Further guidance on the likely properties of the virus strains ofthe invention is provided as follows.

[0087] Preferably, a virus strain of the invention has undergone threeyears or less in culture since isolation of its unmodified clinicalprecursor strain from its host. More preferably, a strain of theinvention has undergone one year or less in culture, for example ninemonths or less, six months or less, three months or less, or two monthsor less, one month or less, two weeks or less, or one week or less. Bythese definitions of time in culture, is meant time actually spent inculture. Thus, for example, it is a common practice to freeze virusstrains in order to preserve them. Evidently, preserving by freezing orin an equivalent manner does not qualify as maintaining the strain inculture. Thus, time spent frozen or otherwise preserved is not includedin the above definitions of time spent in culture. Time spent in cultureis typically time actually spent undergoing serial passage, i.e. timeduring which selection for undesirable characteristics can occur.

[0088] Preferably, a virus strain of the invention has undergone 1,000or less cycles of serial passage since isolation of its unmodifiedclinical precursor strain from its host. More preferably, it hasundergone 500 or less, 100 or less, 90 or less, 80 or less, 70 or less,60 or less, 50 or less, 40 or less, 30 or less, 20 or less, or 10 orless such cycles.

[0089] Preferably, a virus of the invention has a greater ability, asmeasured by standard statistical tests, than a reference laboratorystrain with the equivalent modifications to perform certain functionsuseful in the application at hand. For example, in the case of anoncolytic virus for tumour treatment, a virus strain of the inventionwill preferably have a greater ability than a reference laboratorystrain with equivalent modifications to infect or replicate any tumourcell, to kill tumour cells or to spread between cells in tissue. Morepreferably, such greater ability is a statistically significantlygreater ability. For example, according to the invention, a may have upto 1.1 fold, 1.2 fold, 1.5 fold, 2 fold, 5 fold, 10 fold, 20 fold, 50fold, or 100 fold the capacity of the reference strain in respect of theproperty being tested.

[0090] Preferably, a virus of the invention has, i.e. retains,substantially the ability of its unmodified clinical precursor strain inrespect of one or more of the properties characteristic of usefulness inthe application at hand. For example, in the case of an oncolytic virusintended for the treatment of tumours, a virus strain of the inventionpreferably has substantially the ability of its unmodified clinicalprecursor strain to infect or replicate a tumour cell, kill tumour cellsor to spread between cells in tissue.

[0091] Preferably, according to the invention, a virus retainssubstantially the properties of its unmodified clinical precursor strainif, in a quantitative test, it retains 75%, more preferably 80, 90, 95,98, 99 or 100% of the capacity of the unmodified clinical precursorstrain in respect of the property being tested. More preferably, inrespect of the property being tested, any differences between theunmodified clinical precursor strain and the modified strain of theinvention will not be statistically significant.

[0092] Statistical analysis of the properties described herein may becarried out by standard tests, for example, t-tests, ANOVA, or Chisquared tests. Typically, statistical significance will be measured to alevel of p=0.05 (5%), more preferably p=0.01, p=0.001, p=0.0001,p=0.000001.

[0093] Modifications

[0094] Viruses of the invention are typically modified as compared totheir precursor clinical strains. In particular, certain genes willtypically be rendered non-functional, and the viruses may also comprisea heterologous gene(s). Typically, viruses of the invention areattenuated.

[0095] Viral regions altered for the purposes described herein may beeither eliminated (completely or partly), or made non-functional, orsubstituted by other sequences, in particular by a heterologous genesequence. One or more genes may be rendered non-functional, and one ormore heterologous genes inserted.

[0096] Oncolytic Viruses of the Invention

[0097] In one embodiment, viruses of the invention are modified,oncolytic, non-laboratory viruses. These will be useful in the oncolytictreatment of cancer. Such viruses infect and replicate in tumour cells,subsequently killing the tumour cells. Thus, such viruses arereplication competent. Preferably, they are selectively replicationcompetent in tumour cells. This means that either they replicate intumour cells and not in non-tumour cells, or that they replicate moreeffectively in tumour cells than in non-tumour cells. Measurement ofselective replication competence can be carried out by the testsdescribed herein for measurement of replication and tumour cell-killingcapacity, and also analysed by the statistical techniques mentionedherein if desired.

[0098] An oncolytic virus of the invention preferably has a greaterability than a reference laboratory strain with the same modificationsto infect or replicate in a tumour cell, to kill tumour cells or tospread between cells in tissues. Preferably, this ability is astatistically significantly greater ability as described herein. Theproperties of the virus strain in respect of tumour cells can bemeasured in any manner known in the art.

[0099] For example, the capacity of a virus to infect a tumour cell canbe quantified by measuring the dose of virus required to measure a givenpercentage of cells, for example 50% or 80% of cells. The capacity toreplicate in a tumour cell can be measured by growth measurements suchas those carried out in the Examples (see FIG. 2), e.g. by measuringvirus growth in cells over a period of 6, 12, 24, 36, 48 or 72 hours orlonger.

[0100] The ability of a virus to kill tumour cells can be roughlyquantitated by eye (see FIG. 3) or more exactly quantitated by countingthe number of live cells that remain over time for a given time pointand MOI for given cell type. For example, comparisons may be made over24, 48 or 72 hours and using any known tumour cell type. In particular,HT29 colorectal adenocarcinoma, LNCaP.FGC prostate adenocarcinoma,MDA-MB-231 breast adenocarcinoma, SK-MEL-28 malignant melanoma or U-87MG glioblastoma astrocytoma cells can be used. Any one of these celltypes or any combination of these cell types can be used, as may othertumour cell types. It may be desirable to construct a standard panel oftumour cell types for this purpose. To count the number of live cellsremaining at a given time point, the number of trypan blue-excludingcells (i.e. live cells) can be counted. Quantitation may also be carriedout by fluorescence activated cell sorting (FACS) or MTT assay. Tumourcell-killing ability may also be measured in vivo, e.g. by measuring thereduction in tumour volume engendered by a particular virus.

[0101] The ability of a virus to spread in tissue, especially solidtissue, can be measured by determining the number of cells at sitesconnected to the site of the original infection.

[0102] In order to determine the properties of viruses of the invention,it will generally be desirable to use a standard laboratory referencestrain for comparison. Any suitable standard laboratory reference strainmay be used. In the case of HSV, it is preferred to use one or more ofHSV1 strain 17+, HSV1 strain F or HSV1 strain KOS. The reference strainwill typically have equivalent modifications to the strain of theinvention being tested. Thus, the reference strain will typically haveequivalent modifications, such as gene deletions and/or heterologousgene insertions. For example, in the case of an HSV strain, if theICP34.5 and ICP47-encoding genes have been rendered non-functional inthe virus of the invention, then they will also be renderednon-functional in the reference strain. The modifications made to thereference strain may be identical to those made to the strain of theinvention. By this, it is meant that the gene disruptions in thereference strain will be in exactly equivalent positions to those in thestrain of the invention, e.g. deletions will be of the same size and inthe same place. Similarly, in these embodiments, heterologous genes willbe inserted in the same place, driven by the same promoter, etc.However, it is not essential that identical modifications be made. Whatis important is that the reference gene has functionally equivalentmodifications, e.g. that the same genes are rendered non-functionaland/or the same heterologous gene or genes is inserted.

[0103] In an oncolytic virus of the invention, suitable modificationswill be made to the virus to confer oncolytic activity, if it is notnaturally present, and preferably to confer selective oncolyticactivity.

[0104] In the case of HSV, such mutations allowing selective oncolyticactivity include mutation to the genes encoding ICP34.5, ICP6 and/orthymidine kinase (TK), preferably ICP34.5. Such mutations to theICP34.5-encoding gene in laboratory strains of HSV are described in Chouet al 1990, Maclean et al 1991, although any mutation in which ICP34.5is non-functional may be used.

[0105] Accordingly, in an HSV strain, the viruses preferably modifiedsuch that it lacks one or more of a functional ICP34.5-encoding gene, afunctional ICP6-encoding gene, a functional glycoprotein H-encodinggene, a functional thymidine kinase-encoding gene; or in a non-HSVstrain, the virus lacks a functional gene equivalent to one of said HSVgenes.

[0106] More preferably, the virus lacks a functional ICP34.5-encodinggene.

[0107] Other modifications may also be made. In particular, in the caseof HSV, the virus may be modified such that it lacks a functional ICP47gene. This is because ICP47 usually functions to block antigenpresentation in HSV-infected cells so its disruption leads to a virusthat does not confer on infected tumour cells particular properties thatmight protect such HSV infected cells from the host's immune system.

[0108] Viruses with any other genes deleted/mutated which provideoncolytic properties (ie selective replication in tumours compared tosurrounding tissue) are also viruses of the invention as those skilledin the art will recognise that the above list is not exhaustive andidentification of the function of other genes in any of the virusesabove may suggest the construction of new viruses which are also virusesof the invention.

[0109] Heterologous gene(s) may also be inserted into such viruses ofthe invention by techniques known in the art and/or described herein. Inan oncolytic virus, the heterologous gene will typically be one thatenhances the capacity of the virus to counteract tumours. Any genesconferring on the virus anti-tumour properties may thus be inserted. Inparticular, the heterologous gene may be a gene capable of modifyingimmune response to the tumour cells in a beneficial manner, especiallyan immune stimulatory polypeptide such as CD40L, granulocytemacrophage-colony-stimulating factor (GMCSF), another cytokine orchemokine (e.g.RANTES), B7.1 or B7.2 or IL12. Alternatively, theheterologous gene may encode a pro-drug activator, such asnitroreductase or cytochrome P450. In this context, combined treatmentof tumours with the pro-drug activated by the pro-drug activator and avirus of the invention is envisaged. Alternatively, the heterologousgene may encode a tumour suppressor, such as p53.

[0110] Other Virus Strains of the Invention

[0111] In other embodiments, non-oncolytic viruses are desirable. Thesemay be replication incompetent viruses. Their function is typically todeliver heterologous genes to an individual.

[0112] In particular, for use as a vector in non-oncolytic applications,mutations may be made such that virus regulatory immediate early geneexpression is minimised. Thus, the genes encoding ICP4, ICP27, ICP22and/or ICP0 may be inactivated or deleted either individually or incombination, or mutations in the virion trans-activator protein vmw65included preventing/reducing its trans-activating ability. Inparticularly preferred embodiments for non-oncolytic applications, thegenes encoding ICP27, ICP0 and ICP4 are deleted (with or withoutadditional deletion/inactivation of ICP22 and/or ICP47), or ICP27 andICP4 deleted with an inactivating mutation in vmw65, or ICP4 deleted,again with an inactivating mutation in vmw65. Examples of such virusesinclude viruses reported by Samaniego et al 1998, Krisky et al 1998, orThomas et al 1999.

[0113] Neurotrophic Virus Strains

[0114] Neurotrophic viruses, and particularly herpes simplex virusessuch as HSV1 and HSV2 may be used according to the invention in thedelivery of heterologous genes to the nervous system.

[0115] Viruses according to this embodiment of the invention typicallyhave greater ability than a reference laboratory strain with equivalentmodifications to infect a neuron, spread between cells in nervous tissueor to be transported within an axon. Capacity to infect neurons can bedetermined as set above as for cells in general. Ability to betransported within an axon or to spread between cells in nervous tissuecan be determined by measuring the infection of cells in the nervoussystem at sites connected to the site of the original infection. Resultsmay be analysed statistically as described above.

[0116] In these embodiments, it is preferred that, in an HSV strain, thevirus is modified such that it lacks one, two, three or all of afunctional ICP27-encoding gene, a functional ICP4-encoding gene, afunctional ICP0-encoding gene, or a functional ICP22-encoding gene; or,in an non-HSV strain, the virus lacks a functional gene equivalent toone of said HSV genes; and/or, in an HSV strain, the virus lacks afunctional vmw65 gene due to a mutation in said gene which abolishes itstranscriptional-activation activity; or in a non-HSV strain, the viruslacks a functional gene equivalent to vmw65 due to a mutation in saidgene which abolishes its transcriptional-activation activity.

[0117] Preferably, two or more of the genes encoding ICP27, ICP4, ICP0and ICP22 are rendered non-functional, more preferably three and, stillmore preferably all four. Preferably, according to these embodiments avirus of the invention lacks both a functional gene encoding ICP4 and afunctional gene encoding ICP27 and which has an inactivating mutation inthe gene encoding vmw65 abolishing its transcriptional activationactivity.

[0118] Such viruses may be used for the treatment of either peripheralnervous system disorders or central nervous system disorders. In thecontext of central nervous system disorders, it is particularlypreferred that at least two immediate early genes selected from ICP0,ICP4, ICP22 and ICP27 are rendered non-functional.

[0119] Immunotherapeutic Viruses

[0120] In immunotherapeutic applications, herpes viruses of theinvention are used to infect dendritic cells or other cells of theimmune system, for example macrophages. Normally, herpes virus infectionof dendritic cells reduces the capacity of the dendritic cells tostimulate an immune response. Accordingly, viruses of the invention aremodified so that they are capable of efficiently infecting dendriticcells without preventing the dendritic cells from stimulating the immunesystem (WO00/08191). In such applications, the virus of the inventiontypically comprises a heterologous gene(s)which encodes an antigen geneproduct. This may be an antigen associated with a pathogenic infectionor cancer. The gene is expressed in the dendritic cell and the geneproduce presented as an antigen on the dendritic cell's surface. Thisstimulates an immune response to the antigen by activating T cells,which seek out cells displaying that antigen on their surface, i.e.tumours or pathogen cells or cells infected with a pathogen and destroythem.

[0121] Preferably, in an HSV strain, said virus lacks a functional UL43gene, and/or a functional vhs gene, or, in a non-HSV strain, lacks afunctional equivalent of UL43 and/or vhs; and optionally, in an HSVstrain,, lacks a functional vmw65 gene due to a mutation in said genewhich abolishes its transcriptional-activation activity, or, in anon-HSV strain, lacks a functional gene equivalent to vmw65 due to amutation in said gene which abolishes its transcriptional-activationactivity; and optionally lacks at least one functional immediate earlygene selected from ICP0, ICP4, ICP22 and ICP27.

[0122] In this context, it is preferred that a virus of the inventionhas greater ability than a reference laboratory strain with equivalentmodifications to infect a dendritic cell or to stimulate an immuneresponse. Infection of dendritic cells can be assessed as describedabove for cells in general. In particular, a non-laboratory virus strainof the invention will typically infect a greater percentage of dendriticcells than the reference laboratory strain when the dose of the twostrains is the same. Statistical analysis can be conducted by themethods described above.

[0123] B. Complementing Cell Lines

[0124] When the virus of the invention is a herpes simplex virus whichlacks a particular functional essential gene, for example a geneencoding ICP4 or ICP27, the virus of the invention is propagated on acell line expressing that essential gene. For example, when the viruslacks a functional ICP27 gene, the virus may be propagated on V27 cells(Rice and Knipe, 1990), 2-2 cells (Smith et al, 1992) or B130/2 cells(Howard et al, 1998). When the virus lacks a functional ICP4 gene thevirus may be propagated on a cell line expressing ICP4, for example E5cells (DeLuca et al, 1985). When the virus lacks a functional ICP4 geneand a functional ICP27 gene the virus is propagated on a cell lineexpressing both ICP4 and ICP27 (such as E26 cells; Samaniego et al,1995), and when the virus additionally lacks a functional vmw65 gene thevirus may be propagated on a cell line also containing a non-HSVhomologue of vmw65 (e.g. from equine herpes virus as in Thomas et al,1999). Mutations to vmw65 may also be partially compensated for byinclusion of hexamethylene bisacetamide (HMBA) in the media used forvirus growth (MacFarlane et al, 1992).

[0125] ICP27-expressing cell lines can be produced by co-transfectingmammalian cells, for example the Vero or BHK cells, with a vector,preferably a plasmid vector, comprising a functional HSV ICP27 genecapable of being expressed in said cells, and a vector, preferably aplasmid vector, encoding a selectable marker, for example neomycinresistance. Clones possessing the selectable marker are then screenedfurther to determine which clones also express functional ICP27, forexample on the basis of their ability to support the growth of ICP27−HSV strains, using methods known to those skilled in the art (forexample, as described in Rice and Knipe, 1990).

[0126] Cell lines which do not allow reversion of an ICP27− mutant HSVstrain to a strain with functional ICP27 are produced as describedabove, ensuring that the vector comprising a functional ICP27 gene doesnot contain sequences that overlap with (i.e. are homologous to)sequences remaining in the ICP27− mutant virus.

[0127] Where HSV strains of the invention comprise inactivatingmodifications in other essential genes, for example ICP4, complementingcell lines will comprise a functional HSV gene which complements themodified essential gene in the same manner as described for ICP27. Forexample, in the case of HSV strains comprising mutations in both ICP27and ICP4, a cell line expressing both ICP27 and ICP4 is used (such asdescribed in Samaniego et al, 1995 or in Thomas et al, 1999). HSVstrains expressing other essential genes can be constructed in a similarmanner to that described for ICP27. Here again, if it is ensured thereis no sequence overlap between the remaining virus DNA and that insertedinto the cell line for virus growth, the possibility of reversion of thevirus to a less disabled form during growth will be minimised.

[0128] C. Methods of Mutation

[0129] The various viral genes referred to may be rendered functionallyinactive by several techniques well known in the art. For example, theymay be rendered functionally inactive by deletion(s), substitution(s) orinsertion(s), preferably by deletion. A deletion may remove a portion ofthe genes or the entire gene. For example, deletion of only onenucleotide may be made, resulting in a frame shift. However, preferablya larger deletion is made, for example at least 25%, more preferably atleast 50% of the total coding and non-coding sequence (or alternatively,in absolute terms, at least 10 nucleotides, more preferably at least 100nucleotides, most preferably at least 1000 nucleotides). It isparticularly preferred to remove the entire gene and some of theflanking sequences. An inserted sequence may include one or more of theheterologous genes described below. In the case of the vmw65 gene, theentire gene is not deleted since it encodes an essential structuralprotein, but a small inactivating mutation is made which abolishes theability of vmw65 to activate transcriptionally IE genes (e.g. as in Aceet al, 1989 or Smiley et al, 1997).

[0130] Mutations are made in the herpes viruses by homologousrecombination methods well known to those skilled in the art. Forexample, HSV genomic DNA is transfected together with a vector,preferably a plasmid vector, comprising the mutated sequence flanked byhomologous HSV sequences. The mutated sequence may comprise adeletion(s), insertion(s) or substitution(s), all of which may beconstructed by routine techniques. Insertions may include selectablemarker genes, for example lacZ or GFP, for screening recombinant virusesby, for example β-galactosidase activity or fluorescence.

[0131] D. Heterologous Genes and Promoters

[0132] The viruses of the invention may be modified to carry aheterologous gene/genes. The term “heterologous gene” encompasses anygene. Although a heterologous gene is typically a gene not present inthe genome of a herpes virus, herpes gene/genes may be used providedthat the coding sequence is not operably linked to the viral controlsequences with which it is naturally associated. The heterologous genemay be any allelic variant of a wild-type gene, or it may be a mutantgene. The term “gene” is intended to cover nucleic acid sequences whichare capable of being at least transcribed. Thus, sequences encodingmRNA, tRNA and rRNA are included within this definition. However, thepresent invention is concerned with the expression of polypeptidesrather than tRNA and rRNA. Sequences encoding mRNA will optionallyinclude some or all of 5′ andor 3′ transcribed but untranslated flankingsequences naturally, or otherwise, associated with the translated codingsequence. It may optionally further include the associatedtranscriptional control sequences normally associated with thetranscribed sequences, for example transcriptional stop signals,polyadenylation sites and downstream enhancer elements.

[0133] The heterologous gene/genes may be inserted into the viral genomeby homologous recombination of HSV strains with, for example plasmidvectors carrying the heterologous gene/genes flanked by HSV sequences.The heterologous gene/genes may be introduced into a suitable plasmidvector comprising herpes viral sequences using cloning techniqueswell-known in the art. The heterologous gene/genes may be inserted intothe viral genome at any location provided that the virus can still bepropagated. It is preferred that the heterologous gene/genes is insertedinto an essential gene. Heterologous genes may be inserted at multiplesites within the virus genome.

[0134] The transcribed sequence of the heterologous gene/genes ispreferably operably linked to a control sequence permitting expressionof the heterologous gene/genes in mammalian cells, preferably a tumourcell or a cell of the nervous system. The term “operably linked” refersto a juxtaposition wherein the components described are in arelationship permitting them to function in their intended manner. Acontrol sequence “operably linked” to a coding sequence is ligated insuch a way that expression of the coding sequence is achieved underconditions compatible with the control sequence.

[0135] The control sequence comprises a promoter allowing expression ofthe heterologous gene/genes and a signal for termination oftranscription. The promoter is selected from promoters which arefunctional in mammalian, preferably human, cells of the nervous systemor in tumours or in cells of the immune system. The promoter/promotersmay be derived from promoter sequences of eukaryotic genes. For example,promoters may be derived from the genome of a cell in which expressionof the heterologous gene is to occur, preferably a mammalian, preferablyhuman cell. With respect to eukaryotic promoters, they may be promotersthat function in a ubiquitous manner (such as promoters of β-actin,tubulin) or, alternatively, a tissue-specific manner, such as theneuron-specific enolase (NSE) promoter. They may also be promoters thatrespond to specific stimuli, for example promoters that bind steroidhormone receptors. Viral promoters may also be used, for example theMoloney murine leukaemia virus long terminal repeat (MMLV) LTR promoteror other retroviral promoters, the human or mouse cytomegalovirus (CMV)IE promoter, or promoters of herpes virus genes including those drivingexpression of the latency associated transcripts.

[0136] Expression cassettes and other suitable constructs comprising theheterologous gene/genes and control sequences can be made using routinecloning techniques known to persons skilled in the art (see, for exampleSambrook et al, 1989, Molecular Cloning—A laboratory manual: Cold SpringHarbor Press).

[0137] It may also be advantageous for the promoters to be inducible sothat the levels of expression of the heterologous gene can be regulatedduring the life-time of the cell. Inducible means that the levels ofexpression obtained using the promoter can be regulated. For example, ina preferred embodiment where more than one heterologous gene is insertedinto the HSV genome, one promoter would comprise a promoter responsiveto the tet repressor/VP16 transcriptional activator fusion proteinpreviously reported (Gossen and Bujard, 1992, Grossen et al, 1995), anddriving the heterologous gene the expression of which is to beregulated. The second promoter would comprise a strong promoter (e.g.the CMV IE promoter) driving the expression of the tet repressor/VP16fusion protein. Thus, in this example, expression of the firstheterologous gene would depend on the presence or absence oftetracycline.

[0138] Heterologous genes will typically encode polypeptides oftherapeutic use. For example, in the nervous system in non-oncolyticapplications, genes which may modulate pain, stimulate nerve re-growthor prevent nerve degeneration. In oncolytic applications, heterologousgenes may encode proteins which are themselves cytotoxic, encodepro-drug activating enzymes or which are capable of stimulating orenhancing an anti-tumour immune response.

[0139] Heterologous genes may also include marker genes (for example,encoding β-galactosidase or green fluorescent protein or otherfluorescent proteins) or genes whose products regulate the expression ofother genes (for example, transcriptional regulatory factors includingthe tet repressor/vmw65 transcriptional activator fusion proteindescribed above).

[0140] Gene therapy and other therapeutic applications may well requirethe administration of multiple genes. The expression of multiple genesmay be advantageous for the treatment of a variety of conditions. Herpesviruses are uniquely appropriate as they do not have the limitedpackaging capabilities of other viral vector systems. Thus, multipleheterologous genes can be accommodated within its genome. For example,from 2 to 5 genes may be inserted into the genome.

[0141] There are, for example, at least two ways in which this could beachieved. For example, more than one heterologous gene and associatedcontrol sequences could be introduced into a particular HSV straineither at a single site or at multiple sites in the virus genome. Itwould also be possible to use pairs of promoters (the same or differentpromoters) facing in opposite orientations away from each other, thesepromoters each driving the expression of a heterologous gene (the sameor different heterologous gene) as described above.

[0142] E. Therapeutic Uses

[0143] Viruses of the invention may be used in methods of therapy. Inparticular, oncolytic viruses of the invention may be used inapplications including the oncolytic treatment of cancer, e.g. by directintra-tumour injection. Where the virus comprises a heterologous geneencoding a prodrug activator, additional pro-drug therapy may be carriedout. Additionally, treatment may be combined with or stimulation of animmune response by any means known in the art. Viruses of the inventionmay be used in the therapeutic treatment of any solid tumour in amammal, preferably in a human. For example viruses of the invention maybe administered to a subject with prostate, breast, lung, liver,endometrial, bladder, colon or cervical carcinoma; adenocarcinoma;melanoma; lymphoma; glioma; or sarcomas such as soft tissue and bonesarcomas.

[0144] Replication incompetent viruses of the invention may be used inthe delivery of genes to individuals requiring gene therapy. Inparticular, neurotrophic viruses of the invention may be used in thetreatment of disorders of the central or peripheral nervous system.Preferred central nervous system disorders for treatment or preventioninclude neurodegenerative disorders. Particularly preferred centralnervous system disorders for treatment or prevention are stroke,Parkinson's disease, Alzheimer's disease, Tay Sachs disease andmucopolysaccaride diseases. Preferred peripheral nervous systemdisorders for treatment or prevention include motor neuron disease,chronic pain and peripheral nerve damage.

[0145] Immunotherapeutic viruses of the invention may be used in theprevention or treatment of the pathogenic infection or cancer with whichthe antigen their inserted heterologous coding gene is associated.

[0146] F. Administration

[0147] The viruses of the invention may thus be used in a patient,preferably a human patient, in need of treatment. Viruses of theinvention may be used for the oncolytic treatment of cancer, and herpesviruses of the invention (in addition to oncolytic applications) for thetreatment of, for example pain, degenerative conditions of the nervoussystem, or to stimulate nerve re-growth. The aim of therapeutictreatment is to improve the condition of the patient. Typicallytherapeutic treatment using a virus of the invention will allieviate thesymptoms of the disease or condition of the patient being treated. Amethod of treatment according to the invention therefore comprisesadministering a therapeutically effective amount of a virus of theinvention to a patient suffering from cancer, pain, a neurodegenerativecondition or nerve damage.

[0148] Administration of an oncolytic virus of the invention to apatient suffering from a tumour will typically kill the cells of thetumour thus decreasing the size of the tumour and/or preventing thespread of malignant cells from the tumour. Administration of a virus ofthe invention to a patient suffering from other diseases such as pain,degenerative conditions or nerve damage will typically improve thecondition of the patient. For example by lessening the severity of thepain, slowing the degeneration of nervous tissue or promoting nerveregrowth.

[0149] One method of administering therapy involves combining the viruswith a pharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline.

[0150] Oncolytic treatment and/or gene delivery to cells for therapeuticpurposes may then be carried out following direct injection of thevector composition into target tissue. The amount of virus administeredis in the case of HSV in the range of from 10⁴ to 10¹⁰ pfu, preferablyfrom 10⁵ to 10⁸ pfu, more preferably about 10⁶ to 10⁸ pfu. When injectedfor oncolytic or non-oncolytic treatment, typically up to 500 μl,typically from 1-200 μl, preferably from 1-10 μl of a pharmaceuticalcomposition consisting esentially of the virus and a pharmaceuticallyacceptable suitable carrier or diluent, would be used for injection.However, for some oncolytic therapy applications larger volumes up to 10ml may also be used, depending on the tumour and inoculation site.

[0151] The routes of administration and dosages described are intendedonly as a guide since a skilled practitioner will be able to determinereadily the optimum route of administration and dosage. The dosage maybe determined according to various parameters, especially according tothe age, weight and condition of the patient to be treated, the severityof the disease or condition and the route of administration.

[0152] The preferred route of administration to a patient suffering fromcancer is by direct injection into the tumour. The virus may also beadministered systemically or by injection into a blood vessel supplyingthe tumour. The optimum route of administration will depend on thelocation and size of the tumour.The dosage may be determined accordingto various parameters, especially according to the location of thetumour, the size of the tumour, the age, weight and condition of thepatient to be treated and the route of administration.

[0153] G. Non-Therapeutic Aspects

[0154] Also provided are methods of identifying suitable clinicalstrains for modification according to the invention. In addition,methods of target validation are provided. These concern theidentification of genes suitable for use in the therapeutic applicationsof the invention as described above.

[0155] Methods of production of viruses of the invention are alsoprovided.

[0156] The Following Examples Illustrate the Invention.

[0157] Herpes simplex type-1 virus (HSV1) in which the neurovirulencefactor ICP34.5 is inactivated has previously been shown to direct tumourspecific cell lysis in tumour models both in vitro and in vivo. Suchviruses have also been shown to be safe in Phase I clinical trials bydirect intra-cerebral injection in late stage glioma patients.

[0158] Previous work has used serially passaged laboratory isolates ofHSV1 (viruses derived from HSV1 strain 17+ or HSV1 strain F) which mightbe anticipated to be attenuated in their lytic capability in humantumour cells as compared to more recent clinical isolates.

[0159] In work aimed at producing ICP34.5 deleted HSV with enhancedoncolytic and anti-tumour potential, we have deleted ICP34.5 from anHSV1 clinical isolate and compared replicative and lytic potential in anumber of human tumour cell types in comparison to HSV1 strain 17+ (astandard laboratory strain).

[0160] Virus Construction (See FIG. 1)

[0161] The viruses used were either based on HSV1 strain 17+ (a standardlaboratory strain) or two clinical isolates derived from cold sores fromfrequent re-activators of HSV1. These strains were named BL1 and JS1.ICP34.5 was completely deleted from strain 17+ and JS1 together with theinsertion of a CMV-GFP cassette. JS1 was also further engineered by theinsertion of human or mouse GM-CSF so as to replace the ICP34.5 gene.BL1 and JS1 are thus clinical isolates, or “non-laboratory” strains. Thederivatives of JS1 discussed herein are also non-laboratory strains,i.e. modified non-laboratory strains of the invention.

[0162] Virus Growth in Tumour Cells (See FIG. 2)

[0163] JS1 and BL1 showed enhanced growth in some human tumour cellstested as compared to HSV1 ICP34.5 deleted strain 17+ when tested over a72 hour period (FIG. 2). JS1 was selected for further study and themodifications described above (see FIG. 1, and above) were made to it.

[0164] Lytic Capabilities of Viruses (See FIG. 3)

[0165] Lytic (cell killing) capabilities were enhanced with theJS1-derived non-laboratory strains derived virus in all tumour celllines tested. More particularly, with reference to FIG. 3, the JS1/34.5−virus, i.e. JS1 with ICP34.5 removed by deletion, showed enhanced lyticcapabilities in HT29 colorectal adenocarcinoma, LNCaP.FGC prostateadenocarcinoma, MDA-MB-231 breast adenocarcinoma, SK-MEL-28 malignantmelanoma and U-87 MG glioblastoma astrocytoma cells.

[0166] Lytic capabilities were also assessed in SK-MEL-28, MDA-MB-231and HT29 cells by trypan blue exclusion assay of infected cells atvarious doses and times after infection with BL1, JS1 as compared tostrain 17+. Trypan blue is excluded from live cells and so numbers oflive cells remaining in a culture can be assessed by this means. Tumourcell lines cultured in duplicate wells of six well dishes were infectedfor 24, 48 or 72 hrs at an MOI of 0.1 or 1 with either 17+, BL1 or JS1and numbers of live cells were counted. The percentage of the number oflive cells in equivalent uninfected control wells are shown in Table 1.

[0167] Thus, as in all cases more tumour cells are killed with theclinical isolate viruses BL1 and JS1 than the laboratory isolate 17+, toprovide increased oncolytic activity, the use of recent clinical virusstrains is likely to enhance the anti-tumour capabilities of suchviruses modified to give tumour selective replication (e.g. by thedeletion of ICP34.5) when used in human patients for cancer treatment.TABLE 1 percentage of number of live cells in uninfected control wellstime after JS1 17 BL1 Cell line infection MOI = 0.1 MOI = 1 MOI = 0.1MOI = 1 MOI = 0.1 MOI = 1 SK-MEL-28 24 h 41 8 57.3 19 43.7 6.67duplicate 33.7 7 62.6 19.3 39 6.33 samples 48 h 5.51 1.9 7.4 3.7 4.5 0.85.05 0.8 7.1 2.6 4.8 1.1 72 h 0 0 0 0 0 0 0 0 0 0 0 0 MDA-MB-231 24 h44.91 16.7 69.37 36.34 55.63 26.79 44.02 16.96 65.8 34.55 60.45 25.27 48h 14.1 4.7 27.9 8.3 18 6.7 13.5 3.8 27 8.5 20 8.3 72 h 0 0 2.91 0.731.46 0 0 0 2.91 1.27 1.64 0 HT-29 24 h 37.53 15 47.28 23.61 42.22 22.1539.24 15 45.76 24.24 43.04 21.33 48 h 13.2 2.3 29.4 4.2 18.4 4.4 14 327.7 4.7 21.2 3.7 72 h 0 0 1.57 0 1.64 0 0 0 1.89 0 1.57 0

[0168] Further Enhanced Anti-Tumour Activity

[0169] Further enhanced activity may also be anticipated if theseviruses are then used to deliver genes with anti-tumour activity. Suchgenes include those encoding pro-drug activators or immune stimulatoryproteins.

[0170] For this purpose, we have produced from JS1 an ICP34.5 deletedclinical isolated of HSV1 which expresses human or mouse GM-CSF GM-CSFis a potent immune stimulator. This virus is designed to enhanceanti-tumour immune responses following intra-tumoral injection.

[0171] References

[0172] Chou et al, 1990, Science 250: 1262-1266

[0173] Maclean et al, 1991, J. Gen. Virol. 72: 631-639

[0174] Samaniego et al, 1998, J. Virol. 72: 3307-3320

[0175] Krisky et al, 1998, Gene Therapy 5: 1593-1603

[0176] Thomas et al, 1999, J. Virol. 73: 7399-7409

[0177] MacFarlane et al, 1992, J. Gen. Virol. 73: 285-292

[0178] Howard et al, 1998, Gene Therapy 5: 1137-1147

[0179] Samaniego L A et al, 1995, J. Virol. 69: 5705-5715

[0180] Ace C I et al, 1989, J. Virol. 63: 2260-2269

[0181] Smith I L et al, 1992, Virol. 186: 74-86

[0182] Rice, S A and Knipe D M, 1990, J. Virol. 64: 1704-1715

[0183] DeLuca N A et al, 1985, J. Virol. 56: 558-570

[0184] Gossen M & Bujard H, 1992, PNAS 89: 5547-5551

[0185] Gossen M et al, 1995, Science 268: 1766-1769

[0186] Smiley, J. R. & Duncan J., 1997, J. Virol. 71: 6191-6193

[0187] Thompson et al 1998, Virus Genes 1(3); 275-286

[0188] Meignier et al 1998, J. Infect. Dis. 159; 602-614

[0189] Deposit Information

[0190] HSV1 Strain JS1 has been deposited at the European Collection ofCell Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4 0JG, UnitedKingdom, on Jan. 2, 2001 under provisional accession number 01010209.

1. Use of a modified, oncolytic, non-laboratory virus strain in themanufacture of a medicament for the oncolytic treatment of cancer. 2.Use according to claim 1 wherein said non-laboratory strain: (a) hasundergone one year or less in culture since isolation of its unmodifiedprecursor strain from its host, or (b) has undergone 100 or less cyclesof serial passage since isolation of its unmodified precursor strainfrom its host, or (c) has a greater ability than a reference laboratorystrain with equivalent modifications to infect or replicate in a tumourcell, to kill tumour cells, or to spread between cells in tissue, or (d)has substantially the ability of its unmodified precursor strain inrespect of one or more of the properties defined in (c).
 3. Useaccording to claim 2 wherein, in (c), said greater ability is astatistically significantly greater ability; or wherein, in (d),substantially the ability is the same ability or an ability notstatistically significantly different.
 4. Use according to any one ofthe preceding claims wherein said non-laboratory virus strain is astrain of a herpes virus, adenovirus, picornavirus, retrovirus oralphavirus.
 5. Use according to claim 4 wherein said non-laboratoryvirus strain is a strain of a herpes virus.
 6. Use according to claim 5wherein said non-laboratory virus strain is a strain of a herpes simplexvirus (HSV).
 7. Use according to claim 6 wherein HSV strain is a strainof HSV1 or HSV2.
 8. Use according to any one of claims 5 to 7 wherein,in an HSV strain, the virus is modified such that it lacks one or moreof a functional ICP34.5-encoding gene, a functional ICP6-encoding gene,a functional glycoprotein H-encoding gene, a functional thymidinekinase-encoding gene; or, in a non-HSV strain, the virus lacks afunctional gene equivalent to one of said HSV genes.
 9. Use according toany one of claims 5 to 8 wherein the strain is an HSV strain and thevirus lacks a functional ICP34.5-encoding gene.
 10. Use according toclaim 9 wherein the virus further lacks a functional ICP47 gene.
 11. Useaccording to any one of the preceding claims wherein said non-laboratoryvirus strain further comprises a heterologous gene.
 12. Use according toclaim 11 wherein said heterologous gene is a gene capable of modifyingimmune responses.
 13. Use according to claim 12 wherein saidheterologous gene capable of modifying immune responses encodes animmune stimulatory polypeptide or another gene product capable ofmodifying immune responses, a prodrug activator, a tumour suppressor ora pro-apoptotic gene product.
 14. Use according to claim 13 wherein saidimmune stimulatory polypeptide is granulocyte macrophagecolony-stimulating factor (GMCSF), another cytokine or chemokine,RANTES, B7.1 or B7.2 or IL12, or wherein the prodrug activator isnitroreductase or cytochrome p450, or wherein the tumour suppressor isp53.
 15. Use according to any one claims 2 to 14 wherein thenon-laboratory strain is an HSV strain and the reference strain asdefined in claim 2 is HSV1 strain 17+, HSV1 strain F or HSV1 strain KOSwith equivalent modifications to the non-laboratory strain.
 16. Use of amodified, replication incompetent, non-laboratory virus straincomprising a heterologous gene in the manufacture of a medicament forthe delivery of said gene to a subject.
 17. Use according to claim 16wherein said non-laboratory virus strain: (a) has undergone one year orless in culture since isolation of its unmodified precursor strain fromits host, or (b) has undergone 100 or less cycles of serial passagesince isolation of its unmodified precursor strain from its host, or (c)has a greater ability than a reference laboratory strain with equivalentmodifications to infect a target cell, or (d) has a greater ability thana reference laboratory strain with equivalent modifications, to infect aneuron, to spread between cells in nervous tissue, to be transportedwithin an axon, to infect a dendritic cell or to induce an immuneresponse, or (e) has substantially the ability of its unmodifiedprecursor strain in respect of one or more of the properties defined in(c) or (d).
 18. Use according to claim 17 wherein, in (c), a greaterability is a statistically significantly greater ability; or, in (d),substantially the ability is the same ability or an ability notstatistically significantly different.
 19. Use according to claim 18wherein the non-laboratory virus strain is as defined in any one ofclaims 4 to
 7. 20. Use according to claim 19 wherein in an HSV strain,the virus is modified such that it lacks one, two, three or all of afunctional ICP27-encoding gene, a functional ICP4-encoding gene, afunctional ICP0-encoding gene, or a functional ICP22-encoding gene; or,in an non-HSV strain, the virus lacks a functional gene equivalent toone of said HSV genes; and/or, in an HSV strain, the virus lacks afunctional vmw65 gene due to a mutation in said gene which abolishes itstranscriptional-activation activity; or in a non-HSV strain, the viruslacks a functional gene equivalent to vmw65 due to a mutation in saidgene which abolishes its transcriptional-activation activity.
 21. Useaccording to any one of claims 16 to 20 wherein the medicament is foruse in treating or preventing a peripheral nervous system disorder byadministering said medicament to a peripheral nerve and saidheterologous gene encodes a polypeptide or antisense RNA of therapeuticuse in the treatment of said peripheral nervous system disorder.
 22. Amethod of determining whether a gene has an effect on a phenotypeassociated with a peripheral nervous system disorder or on a cell of theperipheral nervous system which is relevant to a peripheral nervoussystem disorder, which method comprises: (i) inoculating a replicationincompetent herpes virus as defined in any one of claims 16 to 21comprising a heterologous gene into a peripheral nerve; and (ii)monitoring a phenotype of said disorder or an effect of expression ofsaid gene on said cell to determine thereby whether said gene has aneffect relevant to said disorder.
 23. Use according to any one of claims16 to 20 wherein the medicament is for use in treating or preventing acentral nervous system disorder, the heterologous gene encodes apolypeptide or antisense RNA of therapeutic use in treating saiddisorder and the replication incompetent herpes virus comprises: (a) oneor more mutations which prevent or reduce the expression of at least twoimmediate early genes; and (b) a heterologous gene operably linked to apromoter active during herpes virus latency.
 24. Use according to claim27 wherein said strain is an HSV strain and said immediate early geneis, or genes are, one, two, three, or all of the genes encoding ICP0,ICP4, ICP22 and ICP27.
 25. A method of determining whether a gene has aneffect on a phenotype associated with a central nervous system disorderor on a cell of the central nervous system which is relevant to acentral nervous system disorder, which method comprises: (i) inoculatinginto a cell of the central nervous system with a replication incompetentherpes virus as defined in claim 23 or 24; and (ii) monitoring aphenotype of said disorder or an effect of expression of said gene onsaid cell to determine thereby whether said gene has an effect on saidcell or said phenotype.
 26. Use according to any one of claims 16 to 20wherein: the herpes virus is an attenuated herpes virus capable ofefficiently infecting a dendritic cell without preventing the infectedcell from stimulating an immune response; the medicament is for use in amethod of treating or preventing a pathogenic infection or cancer; whichmethod comprises infecting dendritic cells with the said virus; and theheterologous gene encodes an antigen associated with said infection orcancer.
 27. Use according to claim 26 wherein, in an HSV strain, saidvirus lacks a functional UL43 gene, and/or a functional vhs gene, or, ina non-HSV strain, lacks a functional equivalent of UL43 and/or vhs; andoptionally, in an HSV strain, further lacks a functional ICP34.5 gene,or, in a non-HSV strain, a functional equivalent of ICP34.5; andoptionally, in an HSV strain, lacks a functional vmw65 gene due to amutation in said gene which abolishes its transcriptional-activationactivity, or, in a non-HSV strain, lacks a functional gene equivalent tovmw65 due to a mutation in said gene which abolishes itstranscriptional-activation activity; and optionally lacks at least onefunctional immediate early gene.
 28. Use according to claim 27 whereinsaid strain is an HSV strain and said immediate early gene is, or genesare, one, two, three or all of the genes encoding ICP0, ICP4, ICP22 andICP27.
 29. A method of determining whether a gene encodes an antigenassociated with a pathogenic infection or cancer, which method comprisesinfecting a dendritic cell or a macrophage with a virus as defined inany one of claims 26 to 28 and monitoring antigen presentation of thepolypeptide product of said gene, or an effect of expression of saidgene, or a phenotype of said pathogenic infection or cancer to determinethereby whether said gene encodes an antigen associated with saidinfection or cancer and which itself has therapeutic potential or is atarget for therapeutic intervention.
 30. A method of determining thesuitability of a non-laboratory virus strain for modification into amodified strain as defined in any one of the preceding claims,comprising: (i) optionally, isolating a non-laboratory virus strain froma host; (ii) providing said non-laboratory virus strain; (iii) assessingthe properties of the virus in respect of one or more of thecharacteristics defined in part (c) or (d) of claim 2 or part (c), (d)or (e) of claim 17; and, optionally, (iv) selecting for modificationvirus strains with desirable properties.
 31. A method of determining thesuitability of a non-laboratory virus strain for modification into amodified oncolytic strain as defined in any one of claims 1 to 15,comprising: (i) optionally, isolating a non-laboratory virus strain froma host; (ii) assessing the growth of the virus in one or more types oftumour cell; and optionally (iii) selecting for modification virusstrains with a high growth rate.
 32. A method according to claim 31wherein the virus is as defined in any one of claims 4 to
 7. 33. Amethod of determining the suitability of a non-laboratory virus strainfor modification into a modified oncolytic strain, comprising: (i)providing a non-laboratory virus strain, optionally one selected by amethod according to step (ii) of claim 31 or 32; (ii) modifying saidstrain such that it becomes oncolytic; and (iii) assessing the abilityof said modified, oncolytic non-laboratory strain to kill tumour cells;and optionally (iv) selecting strains that exhibit high tumourcell-killing capacity for further modifications; and optionally (v)carrying out further modifications.
 34. A method according to claim 33wherein said virus is as defined in any one of claims 4 to
 7. 35. Amethod according to claim 33 wherein the virus is a herpes virus and, inan HSV strain, it is modified by rendering non-functional one or moregenes selected from the genes encoding ICP34.5, ICP6 and thymidinekinase; or, in a non-HSV strain, it is modified by renderingnon-functional a gene or genes equivalent to said HSV gene or genes. 36.A method according to any one of claims 33 to 35 wherein the tumourcell-killing capacity of said modified, oncolytic non-laboratory strainis measured by comparison to the tumour cell-killing capacity of areference laboratory strain with the same gene or genes renderednon-functional.
 37. A method according to claim 36 wherein thenon-laboratory strain is an HSV strain and the reference stain is HSV1strain 17+, HSV strain F or HSV strain KOS.
 38. A method of determiningwhether a gene enhances the anti-tumour effects of a virus comprising:(i) providing a modified, oncolytic non-laboratory strain as defined inany one of claims 1 to 15; (ii) inserting said gene into said virus as aheterologous gene; and (iii) assessing the ability of said modified,oncolytic non-laboratory strain to kill tumour cells compared to theability of the precursor strain provided in step (i).
 39. A method ofproducing a modified, oncolytic, non-laboratory virus strain comprising:(i) isolating from a host a non-laboratory strain of a virus; (ii)optionally determining its suitability for modification as set out inclaim 33; and (iii) modifying it to render it oncolytic, and optionally(iv) carrying out further modifications.
 40. A method according to claim39 wherein the resulting modified, oncolytic, non-laboratory virus is asdefined in any one of claims 2 to
 15. 41. A method of producing amodified non-laboratory virus strain comprising: (i) providing anon-laboratory strain of a virus; (ii) modifying it to render itreplication incompetent and, (iii) inserting a heterologous gene.
 42. Amethod according to claim 41 wherein the resulting modifiednon-laboratory virus strain is as defined in any one of claims 16 to 29.43. A modified, oncolytic, non-laboratory virus strain as defined in anyone of claims 1 to
 15. 44. A modified non-laboratory virus straincomprising a heterologous gene, as defined in any one of claims 16 to29.
 45. A modified non-laboratory virus strain according to claim 43 or44 which is a herpes virus strain.
 46. A modified non-laboratory virusstrain according to claim 45 which is a herpes simplex virus (HSV)strain.
 47. Use of a gene identified as having an effect on a phenotypeassociated with a peripheral nervous system disorder or on a cell of theperipheral nervous system which is relevant to a peripheral nervoussystem disorder by a method according to claim 22, or of a gene productencoded by said gene in the manufacture of a medicament for thetreatment of a peripheral nervous system disorder.
 48. Use of a geneidentified as having an effect on a phenotype associated with a centralnervous system disorder or on a cell of the central nervous system whichis relevant to a central nervous system disorder by a method accordingto claim 23, or of a gene product encoded by said gene in themanufacture of a medicament for the treatment of a central nervoussystem disorder.
 49. Use of a gene identified as encoding an antigenassociated with a pathogenic infection or cancer by a method accordingto claim 29, or of an antigen encoded by said gene in the manufacture ofa medicament for the treatment or prevention of said infection orcancer.
 50. A non-laboratory virus strain identified by, or produced inthe course of, a method according to any one of claims 30 to
 42. 51. Useof a gene identified as enhancing the anti-tumour effects of a virus bya method according to claim 38, or of a gene product encoded by saidgene, in the manufacture of a medicament for the treatment or preventionof cancer.
 52. A modified non-laboratory virus strain obtained orobtainable by a method according to any one of claims 39 to
 42. 53. HSV1strain JS1 as deposited at the European Collection of Cell Cultures(ECACC) under provisional accession number 01010209, or an HSV1 strainderived therefrom.
 54. An HSV1 strain derived from JS1 as defined inclaim 53 and having the characteristics set out in any one of claims 2to
 29. 55. An HSV1 strain derived from JS1 as defined in claim 54 andmodified such that it lacks a functional ICP34.5 gene, and optionally afunctional ICP47 gene; and optionally further comprises a heterologousgene encoding GMCSF.
 56. A pharmaceutical composition comprising a virusas defined in any one of claims 53 to
 55. 57. A virus as defined in anyone of claims 53 to 55 for use in the treatment of the human or animalbody.
 58. A method of treating a tumour in an individual in need thereofby administering to said individual an effective amount of a virus asdefined in claim
 1. 59. A method of delivering a gene to an individualin need thereof by administering to said individual an effective amountof a virus as defined in claim
 16. 60. A method of treating orpreventing a peripheral nervous system disorder by administering to aperipheral nerve of an individual in need thereof an effective amount ofa virus as defined in claim
 21. 61. A method of treating or preventing acentral nervous system disorder comprising administering to anindividual in need thereof a virus as defined in claim 23.